Low-Field NMR Analyses of Gels and Starch-Stabilized Emulsions with Modiﬁed Potato Starches

: Many different biopolymers are used to stabilize emulsions, of which starch is of particular concern. To improve the characteristics and technical utility of native starch, various types of changes can be made. This article is a report describing the molecular dynamics of water by the low-ﬁeld nuclear magnetic resonance (LF NMR) of chemically (E 1412 and E 1420) and physically modiﬁed starch (LU 1432) gels and the effect of their use on the stability of oil/water emulsions obtained using bovine and porcine fats. The analysis of changes in spin–spin and spin–lattice relaxation times over time showed that the presence of the type of starch modiﬁcation signiﬁcantly affects the values of T 1 and T 2 relaxation times, as well as the correlation times. Research on time-related changes in water binding in oil-in-water emulsions showed that potato starch modiﬁed by chemical methods can be used as an emulsiﬁer. Compared to physically modiﬁed starch, chemically modiﬁed starches have a much better water-binding capacity.


Introduction
Starch, one of the two most abundant biopolymers in nature, is produced by many plants as an energy storage compound. It consists of two fractions-linear amylose composed of anhydroglucose units linked by α-1,4-glycosidic bonds and branched amylopectin containing additional α-1,6-glycosidic bonds [1,2].
Natural starch, although widely used in many areas of life for years, has some disadvantages that affect its functionality. The main disadvantage of native starch is the instability of the paste it creates [3]. The viscosity of the starch solution may change under the influence of temperature, mechanical forces (mixing), changes in the reaction of the environment, and pressure. Clear starch solutions, after cooling down and forming a gel, quickly retrograde, which is externally evident by the clouding of the paste [4,5]. Various modifications are used to eliminate these drawbacks. They are intended to improve certain functional properties or to impart new properties, specific to a given application. On the industrial scale, starches are modified with physical, chemical, and enzymatic methods or combinations of these [6][7][8].
Modifications of starch are aimed at giving it new physicochemical and functional properties not present in native starches. As a result of these modifications, the obtained modified starches differ significantly from natural starches in at least one property. This mainly applies to rheological properties [9,10] such as the binding of large amounts of water with substances dissolved in it; viscosity; gelatinization temperature; formation of various types of gels; not yielding to or being slightly retrograde; clarity and transparency of pastes and gels; absorption of various compounds; or the ability to emulsify lipophilic compounds in water and to stabilize emulsions and suspensions.
Starch is widely used in many industries, whether it serves as a filler, binder, or glue [11][12][13][14]. In the food industry, native starch is often used as a thickening agent or stabilizer. In addition, it has also found wide application in the production technologies of meat preparations, especially as a good substitute for fats in low-calorie products [15].
The growing demand for low-fat meat products prompts producers to modify their existing recipes. Starch is frequently used to partially substitute fat in meat products [16].
In the presence of a sufficiently large amount of water and during heating, the starch granules lose their native structure, and the system takes the form of a sol. As the starch system cools in the water, the gelatinization process takes place [17]. Therefore, it seems interesting to understand the mechanisms of the interaction of animal fat and starch during the gelatinization process.
In the 1990s, low-field nuclear magnetic resonance became a method for studying many biological systems [18][19][20], such as food or model biopolymers [21][22][23][24][25]. In contrast to other analytical methods of biopolymer gel characterization, LF NMR is a technique that does not destroy the samples analyzed, and therefore it is possible to perform multiple measurements on the same system, e.g., examine the retrogradation processes or changes in water behavior during storage [26]. The tests consist in measuring spin-lattice T 1 relaxation times or spin-spin T 2 relaxation times or the analysis of the free induction delay shape [26,27]. Due to the use of magnetic fields, the induction value of which (B 0 ) does not exceed 1 Tesla, this method is very well suited for studying the interactions between water molecules and other macromolecules in the system. It is therefore a good method of analyzing biopolymer gels. The LF NMR method enables the analysis of the molecular dynamics of water, which is a solvent, i.e., the liquid phase of a biopolymer gel [28,29]. The processes of proton relaxation in such systems are, compared to the state of liquid water, slowed down due to the presence of biomolecules forming a spatial lattice. The use of this method complements the knowledge of biopolymer systems at the molecular level. A detailed analysis of changes in the molecular dynamics of water in biopolymer gels may also be an important contribution to understanding the issues of starch retrogradation at the molecular level [30].
Considering the above, research was undertaken to track changes in the molecular dynamics of protons in the systems of oil-in-water (O/W)-type emulsions and to analyze the impact of the presence of potato starch modified with various methods (physically and chemically) on the stability of such systems.

Materials
The tested materials were two chemically modified potato starches (E 1420 and E 1412) and one physically modified (LU 1432). Ready, commercial modified starches were purchased from WPPZ S.A. (Luboń, Poland). Fats (bovine and porcine) were obtained from Animex Foods sp. z o.o. (Morliny, Poland).

Preparation of Gels
The prepared starch suspensions in water (0.25 g/g) were subjected to thermal treatment (90 • C) in a water bath for 1 h with constant stirring. The obtained starch gels were then placed in glass tubes and sealed using Parafilm ® . LF NMR measurements were taken for 8 h at 1 h intervals. Between the measurements, the samples were stored in a refrigerator (4 • C), and before the next measurements, the samples were heated in the spectrometer head to the measurement temperature (25 • C).

Preparation of Emulsions
O/W emulsions were prepared according to the method described earlier by Małyszek et al. [31]. Emulsions containing 1 part starch, 1 part fat, and 3 parts water were analyzed. Briefly, the tested fats and water, before mixing with starch, were heated to a temperature of 40 • C, and then, after adding starch, the sample was heated to 90 • C and thermostated Processes 2022, 10, 2109 3 of 14 for 1 h with continuous stirring using a mechanical stirrer MS-H-Pro (ChemLab, Kielce, Poland) at 300 rpm. Freshly prepared samples were placed in NMR tubes, sealed with Parafilm ® , and placed in an NMR spectrometer.

1 H NMR Relaxometry
Measurements of spin-lattice T 1 and spin-spin T 2 relaxation times were carried out using a pulsed NMR spectrometer (Ellab, Poznań, Poland) equipped with an integral temperature control system. The device was operating at a frequency of 15 MHz at the temperature of 25.0 • C ± 0.5 • C. For T 1 measurements, the inversion-recovery pulse sequence (π−TI−π/2) was used [27], and the distances between pulses (TI) were varied from 2 to 2000 ms. Each time, 32 sequences of pulses were generated, and 119 points were collected on each signal of free induction decay (FID) recorded after the π/2 pulse. The repetition time between successive pulse sequences was 15 s. The values of T 1 were calculated with the use of the CracSpin program [32], in which the Marquardt method enables minimization by fitting to the multi-exponential course of the magnetization regrowth curve. The regrowth of the longitudinal component of magnetization (M z ) is described by the formula (Equation (1)): where M 0 -equilibrium value of magnetization, p i -fraction of protons relaxing with time T 1i . For T 2 measurements, a CPMG impulse sequence (π/2−TE−π n ) was used [33,34]. The distance between the spin echoes (2TE) was 3 ms. The 5 accumulation signals were applied in a spin echo sequence. The decay of the spin echo amplitudes (M x,y ) can be expressed by the formula (Equation (2)): where p i -fraction of protons relaxing with time T 2i .
The calculation of the relaxation time was performed with the adjustment of the recorded delays of the spin echo amplitudes to the formula taking into account the multiexponential delay.
The relaxation time values were used to calculate the correlation times (τ c ), in accordance with Bloembergen-Purcell-Pound formulas [35] (Equations (2) and (3)): where µ 0 is the permittivity of free space, γ is the magnetogyric ratio, h is the Planck constant, r 0 is the distance of the interacting nuclei, and ω is the resonance frequency (ω = 2πf , f is the Larmor frequency, the spectrometer frequency).

Relaxation of Starch Gels
In the first step, the molecular dynamics of water in modified starch gels were analyzed. The time value T 1 corresponds to the bulk water content, while the value T 2 describes the mobility of water molecules [26,27]. The starch concentration was constant and amounted to 0.25 g/g in each sample. All gels had one spin-lattice (T 1 ) relaxation time and one spin-spin (T 2 ) relaxation time (Table 1). This reflects a rapid chemical energy exchange between the protons of the molecules of water hydrating the biopolymer and the molecules of water located in the spaces in the polymer network [36]. Table 1. Values of spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation times in modified potato starch gels.

Time (h)
E 1420 E 1412 LU 1432 Changes in the values of the correlation times in the starch gels tested over time are shown in Figure 1. This parameter characterizes the rotational movement of molecules containing protons; thus, it describes the freedom of molecular movement in the analyzed samples [37]. In the gels of chemically modified starch, a significant increase in the values of the correlation times is observed up to about 3 h from the moment the gel reaches the ambient temperature; then, the values of τ c decrease. A similar course of τ c was recorded for native starch gels [38]. This means that water is first bound in the structure of the starch gel, and then it is removed inside the network. The recorded lower values of mean correlation times for E 1420 starch gels, compared to E 1412, mean that the water in this system is bound less strongly. All analyzed starches are characterized by changes in the values of mean correlation times in the time interval from about 1 to 6 h; however, it was found that these changes were much smaller in the case of LU 1432 starch gels. It can therefore be concluded that the physically modified potato starch forms gels with a structure more stable over time, and the changes in water binding in such gels are small, which was also demonstrated in the studies described by Małyszek et al. [31].

Relaxation of O/W Emulsions
In order to prepare a stable emulsion, it is necessary to use an additional substance that reduces the interfacial tension at the oil/water interface. Reducing the fat content in the absence of technological additives results in free water in the system [39]. For this reason, in order to obtain a stable emulsion with a reduced fat content, it is necessary to use emulsifying substances. Starch does not have the ability to do this; nevertheless, it acts as an emulsion stabilizer, effectively increasing the stability of the two-phase system [40]. Emulsifiers are substances that promote the formation of emulsions and stabilize them for a short time due to interfacial interaction. On the other hand, the role of emulsion stabilizers is attributed to polysaccharides or, more broadly, to hydrocolloids, mainly through the mechanism of increasing the viscosity of the dispersion phase. Long-term stability of the emulsion is thus provided, possibly due to the adsorption mechanism [40][41][42]. Native starch can also be used as an emulsion stabilizer, but it is not the most effective in this role. This is related to surface activity, which is much better in both physically and chemically modified starches. Modifications may improve the emulsifying capacity of starch, an example being OSA starch (E 1450) [43].

Relaxation of O/W Emulsions
In order to prepare a stable emulsion, it is necessary to use an additional substa that reduces the interfacial tension at the oil/water interface. Reducing the fat conten the absence of technological additives results in free water in the system [39]. For reason, in order to obtain a stable emulsion with a reduced fat content, it is necessary use emulsifying substances. Starch does not have the ability to do this; nevertheless, it a as an emulsion stabilizer, effectively increasing the stability of the two-phase system [ Emulsifiers are substances that promote the formation of emulsions and stabilize them a short time due to interfacial interaction. On the other hand, the role of emulsion sta lizers is attributed to polysaccharides or, more broadly, to hydrocolloids, mainly throu the mechanism of increasing the viscosity of the dispersion phase. Long-term stability the emulsion is thus provided, possibly due to the adsorption mechanism [40][41][42]. Nat starch can also be used as an emulsion stabilizer, but it is not the most effective in this r This is related to surface activity, which is much better in both physically and chemic modified starches. Modifications may improve the emulsifying capacity of starch, an ample being OSA starch (E 1450) [43].
The changes in water binding in O/W emulsions stabilized with modified pot starches were analyzed up to 8 h. All emulsions contained three parts water, one part and one part starch, so the starch concentration was always 0.25 g/g. The results of relaxation times measurements are shown in Table 2. As in the case of O/W emulsi emulsified with native starch [38], two proton fractions were found in the emulsions em sified with modified starches. Analyses of O/W emulsions with the addition of starch vealed two components of spin-lattice T1 and spin-spin T2 relaxation times, in line w published data [38]. This means that there were two proton fractions relaxing at differ rates in the system and that the energy exchange between these proton fractions was m slower than the relaxation time [44]. The analysis of the values of both relaxation ti components indicates that the long components (T12 and T22) reflected the relaxation p cesses of proton fractions associated mainly with the biopolymer gel, and the short co ponents of the relaxation times (T11 and T21) were characterized by much lower valu The changes in water binding in O/W emulsions stabilized with modified potato starches were analyzed up to 8 h. All emulsions contained three parts water, one part fat, and one part starch, so the starch concentration was always 0.25 g/g. The results of the relaxation times measurements are shown in Table 2. As in the case of O/W emulsions emulsified with native starch [38], two proton fractions were found in the emulsions emulsified with modified starches. Analyses of O/W emulsions with the addition of starch revealed two components of spin-lattice T 1 and spin-spin T 2 relaxation times, in line with published data [38]. This means that there were two proton fractions relaxing at different rates in the system and that the energy exchange between these proton fractions was much slower than the relaxation time [44]. The analysis of the values of both relaxation time components indicates that the long components (T 12 and T 22 ) reflected the relaxation processes of proton fractions associated mainly with the biopolymer gel, and the short components of the relaxation times (T 11 and T 21 ) were characterized by much lower values, which suggests that these components describe relaxation of the proton fraction of the fat emulsion. Therefore, the changes in long and short components of relaxation times in the studied emulsions were analyzed separately.
The time-related changes in the mean values of the correlation times of the mobility of water molecules were determined on the basis of the values of the relaxation times and are shown in Figure 2A 0  37  30  439  371  48  39  440  147  1  85  30  402  389  40  30  427  122  2  90  28  398  129  52  40  432  127  3  80  35  402  384  55  31  427  133  4  80  33  414  397  58  36  425  147  5  77  30  401  399  76  32  421  186  6  73  26  404  285  55  34  422  152  7  68  31  405  203  29  35  428  100  8  67  26  396  207  34  37  431  107 Literature data confirm that physically modified starch, despite its low surface activity, can be successfully used to create an O/W emulsion [45]. In the emulsions emulsified with starch LU 1432, changes were found depending on the applied fat. In the case of porcine fat, the fraction of fatty protons is less mobile than the fraction of starch protons. The opposite relationship was observed for bovine fat, where the fraction of fatty protons is definitely more mobile than the starch fraction.
Measurements of water binding in gels and O/W emulsions were also carried out in long-term storage. The tests were carried out up to 6 days from the moment of preparation of the emulsion, and the emulsions were stored at a temperature of 4 • C in the intervals between measurements. The values of relaxation times for starch gels are summarized in Table 3. The obtained results show that E 1420 and E 1412 starches show similar emulsifying properties due to the water-binding properties.
Literature data confirm that physically modified starch, despite its low surface activity, can be successfully used to create an O/W emulsion [45]. In the emulsions emulsified with starch LU 1432, changes were found depending on the applied fat. In the case of porcine fat, the fraction of fatty protons is less mobile than the fraction of starch protons. The opposite relationship was observed for bovine fat, where the fraction of fatty protons is definitely more mobile than the starch fraction.   The values of relaxation times presented in the table indicate differences in T 1 values. The gels of unmodified potato starch (see [38]) and E 1421 starch show longer spin-lattice relaxation times than the E 1412 and LU 1432 starch gels, which suggests that E 1412 and LU 1432 bind water better. Such properties of starch gels may positively affect the long-term stability of food products in which these starches are used, suggesting their beneficial properties as food additives [46].
The changes in water binding of modified starches were analyzed by changes in the mean values of proton motion correlation times in the tested gels, and the obtained results are presented in Figure 3.
The changes in water binding of modified starches were analyzed by changes in mean values of proton motion correlation times in the tested gels, and the obtained resu are presented in Figure 3.
storage time (h) 20 40 60 80 100 120 140 mean correlation time (s) x10 -8  The obtained results show that LU 1432 starch gels are characterized by the short mean values of proton motion correlation times, which means that this starch binds wa the least. On the other hand, E 1420 starch forms a gel with the strongest binding of wat which was also proven by other authors [47]. Gels made of modified starches E 1412 a The obtained results show that LU 1432 starch gels are characterized by the shortest mean values of proton motion correlation times, which means that this starch binds water the least. On the other hand, E 1420 starch forms a gel with the strongest binding of water, which was also proven by other authors [47]. Gels made of modified starches E 1412 and LU 1432 show minimum τ c values after about 2 days, which proves the initial release of water molecules by the network. After this time, an increase in the value of correlation times means a slow binding of water molecules outside the polymer network. Time-dependent changes in the relaxation times of the protons of starch and fat fractions in O/W emulsions emulsified with modified starches are summarized in Table 4. Based on the results of the relaxation times, the mean correlation times of protons in these fractions were calculated. Time-dependent changes in the values of correlation times allowed us to conclude about water binding in these systems. Fat proton fractions (T 11 and T 21 ) in porcine and bovine fat emulsions are shown in Figures 4 and 5. In porcine fat emulsions, changes in the dynamics of water molecules are observed for up to 6 days, with the greatest changes being noted on days 2 to 4. After this time, however, all tested emulsions show similar correlation times.
In emulsions based on bovine fat, time-related changes in the molecular dynamics of the fat fraction show large differences due to the starch used in the emulsion. The lowest values of correlation times were found in the systems emulsified with LU 1432 starch. In comparison to emulsions with porcine fat, differences in the values of correlation times after 6 days of storage were found in emulsions with bovine fat. Figures 6 and 7 show the time-dependent changes in the mean correlation times in the starch proton fractions (T 12 and T 22 ) of the emulsion.
The use of LU 1432 starch as an emulsifier made the starch fraction of protons in the porcine fat emulsion stable in time. The mean values of proton motion correlation times are the smallest, which reflects the weakest binding of water compared to the other analyzed starches. The emulsions with E 1420 starch are also time-stable, but in this system, the correlation times are much longer compared to the LU 1432 starch-stabilized emulsions. This means that E 1420 starch is an emulsifier that inhibits molecular movements of protons in the starch fraction of the emulsion. The best water binding, already after about 3 days of storage, was found when using starch E 1412 as an emulsifier in porcine fat emulsions.      The use of LU 1432 starch as an emulsifier made the starch fraction of protons in porcine fat emulsion stable in time. The mean values of proton motion correlation tim are the smallest, which reflects the weakest binding of water compared to the other a lyzed starches. The emulsions with E 1420 starch are also time-stable, but in this syste the correlation times are much longer compared to the LU 1432 starch-stabilized em sions. This means that E 1420 starch is an emulsifier that inhibits molecular movement protons in the starch fraction of the emulsion. The best water binding, already after ab 3 days of storage, was found when using starch E 1412 as an emulsifier in porcine emulsions. Analysis of animal fat emulsions stabilized with chemically modified starches (E 14 and E 1412) showed that these emulsions are characterized by the relatively low dynam of water molecules. This suggests a favorable, especially long-term, water binding in presence of animal fats [47,48]. However, the results obtained suggest that the LU 14 starch, which was only subjected to thermal treatment, does not fulfill the role of an em sifier well. The water binding of physically modified starch is much weaker than that chemically modified starches [49]. Differences in time changes of the mean values of p ton motion correlation times are observed depending on the type of fat used. Bovine emulsions can be successfully stabilized with E 1420 starch. On the other hand, porc fat emulsions stabilized with E 1412 starch are characterized by better water bindi Analysis of animal fat emulsions stabilized with chemically modified starches (E 1420 and E 1412) showed that these emulsions are characterized by the relatively low dynamics of water molecules. This suggests a favorable, especially long-term, water binding in the presence of animal fats [47,48]. However, the results obtained suggest that the LU 1432 starch, which was only subjected to thermal treatment, does not fulfill the role of an emulsifier well. The water binding of physically modified starch is much weaker than that of chemically modified starches [49]. Differences in time changes of the mean values of proton motion correlation times are observed depending on the type of fat used. Bovine fat emulsions can be successfully stabilized with E 1420 starch. On the other hand, porcine fat emulsions stabilized with E 1412 starch are characterized by better water binding. Thus, when using porcine fat in an emulsion, E 1412 starch can be recommended as an emulsifier or a substitute for porcine fat. In their research, Yang et al. [50] showed the beneficial effects of two different emulsifiers, so it can be assumed that it is also possible to obtain a synergistic effect when using different starches. Further research into the interactions of different starches could deepen our understanding of the mechanisms underlying emulsion formation and stabilization observed in current experiments.

Conclusions
To the best of our knowledge, this report is the first molecular-level description of the effect of the type of starch modification on the long-term stability of gels and emulsions obtained from animal fat. On the basis of the obtained results, it was found that chemically modified starch stabilizes the O/W emulsion significantly better than the physically modified starch. Nevertheless, depending on the type of fat, different modified starches appear to be more effective. For stabilizing the bovine-fat-based emulsions, smaller changes in stability were noted with starch E 1420, while the stabilizing effect in porcinefat-based emulsions was stronger with E 1412.